Modern microbiological diagnostic assays employ two different growth protocols for analysis, i.e., detection, identification and/or enumeration, of microorganisms: (1) analysis without preliminary growth of the microorganisms or (2) analysis after preliminary growth of microorganisms. Analysis of microorganisms without preliminary growth includes the use of methods such as: (1) immunological analyses, e.g., immunofluorescence, radioimmunoassay, enzyme immunoassay (EIA) for single cells and others; (2) DNA/RNA analyses via polymerase chain reaction (PCR); and (3) flow cytometry (FC) analyses (detection of single cells after labeling with fluorescent antibodies or fluorogenic substrates). Artificial substrates may also be used for detection and analysis of microorganisms by microscopic means. Microbiological diagnostic assays that analyze microorganisms after preliminary growth of the microorganisms include enzyme-linked immunosorbent assay (ELISA), mass-spectrometry, Fourier transform infrared (FTIR) spectroscopy, immune analyses and others.
Whether one chooses to use diagnostic assays with or without preliminary growth of microorganisms, all of the current diagnostic assays are cost, time and labor intensive and require the use of sophisticated laboratory equipment and personnel.
Along with the above-described sophisticated techniques, the traditional method of colony growth on a Petri plate still is the most common method used to detect live microorganisms in a sample. However, analysis by Petri plate also can be time and labor intensive. In order to create colonies, inoculated plates typically are incubated approximately 24 to 48 hours for bacteria and 72 to 120 hours for fungi. Thus, a relatively long time is needed to form colonies easily visible to the naked eye. If the sample arises from a time-sensitive biohazard incident, a hospital patient in critical condition, or industrial (food, pharmaceutical) products having a short shelf life, then time is of the essence and time-consuming incubation and serial testing can be a substantial burden with potentially life-threatening or profit loss consequences. Colonies appearing on solid nutrient media typically are counted for detection and enumeration of total microbial growth or are removed and analyzed according to traditional microbiological procedures, e.g., mass-spectrometry, Fourier transform infrared spectroscopy (FTIR) spectroscopy, chromatography, immunoassays or PCR.
In the field of medicine, in the food, biotechnological and pharmaceutical industries, for military and civilian defense, and environmental control, it is very important to have rapid, simple and reliable identification of colonies and microcolonies. For example, pathogens which affect the food industry are E. coli, Salmonella spp., Listeria spp., Pseudomonas aeruginosa, Staphylococcus aureus, some Lactobacillus spp., Bacillus cereus, some yeast and molds and other microorganisms. Pathogens which require rapid identification for military and civilian defense include, for example, anthrax, where time is of the essence. Reliable monoclonal and polyclonal antibodies exist for a majority of these organisms but they are most useful only after a high quantity of cells are grown on a special media or broth and then identified by EIA or ELISA using sophisticated and expensive equipment such as VITEC™ (bioMerieux) by fluorescence or using 96-well plates with specific antibodies immobilized inside the wells.
Consequently, there is a need for a rapid, accurate and cost-effective method to selectively detect and identify live microorganisms which overcomes the limitations of the prior art.